Synthetic hla binding peptide analogues and uses thereof

ABSTRACT

The present invention is directed to a synthetic peptide comprising a sequence of amino acids containing at least a segment that is an analogue of a native peptide that specifically binds to HLA A0201 or HLA A0301 molecules on a cell characteristic of a pathophysiologic state in a mammal. The synthetic peptide may be derived form native peptides comprising a breakpoint region of the WT1 protein.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/223,139, filed Sep. 12, 2005, issued as U.S. Pat. No. 7,598,221,which is a continuation of Ser. No. 10/999,425, filed Nov. 30, 2004,issued as U.S. Pat. No. 7,488,718, which claims benefit of priority ofU.S. Provisional Application Ser. No. 60/525,955, filed Dec. 1, 2003.These applications are hereby incorporated in their entirety byreference herein.

This invention was produced in part using funds obtained throughNational Cancer Institute Core Grant No. 08748 and National Institutesof Health Grant Nos. PO1 33049 and PO1 2376, consequently, the federalgovernment has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to the fields of immunology and leukemia therapy.More specifically, this invention relates to the use of syntheticanalogue peptides to induce heteroclitic human T cell responses againstnative peptides of the synthetic analogues.

DESCRIPTION OF THE RELATED ART

Chronic myelogenous leukemia (CML) is a pluripotent stem cell disordercharacterized by the presence of the Philadelphia chromosome (Ph). ThePhiladelphia chromosome represents a translocation in which the c-abloncogene has moved from chromosome 9 into the breakpoint cluster region(bcr) within the bcr gene on chromosome 22, resulting in a chimericbcr-abl gene. The fused genes encode an 8.5 kb chimeric mRNA which isusually translated into a 210-kDa or 190-kDa protein. This bcr-ablprotein is a tyrosine kinase which is uniquely present in the leukemiacells of chronic myelogenous leukemia patients and is necessary andsufficient for transformation. In chronic myelogenous leukemia, thebreakpoint in the bcr gene occurs either between bcr exon 2 (b2) and 3(b3) or between bcr exon 3(b3) and 4(b4). Although aberrant bcr-ablfusion genes and bcr-abl mRNA splicing can occur, the majority ofpatients with chronic myelogenous leukemia therefore express p210-b.3a2or p.210-b2a2; often both p210 and pi90 proteins are expressed togetherwith low levels of p190-ela2 bcr-abl proteins. In Phi positive acutelymphocytic leukemia (ALL), the predominant breakpoint is at the ela2site.

The chimeric fusion proteins are potential antigens. First, the proteinsare uniquely expressed in chronic myelogenous leukemia cells in whichthe functional regions contain a sequence of amino acids that is notexpressed on any normal protein. Secondly, as a result of the codonsplit on the fused message, a new amino acid, lysine in b3a2, and aconserved amino acid, glutamic acid in b2a2, is present at the exactfusion point in each of the proteins. Therefore, the unique amino acidsequences encompassing the b3a2 and b2a.2 breakpoint region can beconsidered truly tumor specific antigens. Despite the intracellularlocation of these proteins, short peptides produced by cellularprocessing of the products of the fusion proteins can be presented onthe cell surface within the cleft of HLA molecules and in this form theycan be recognized by T cells.

Recent clinical trials demonstrated that a rumor specific, bcr-ablderived multivalent vaccine may be safely administered to patients withchronic phase chronic myelogenous leukemia The vaccine reliably elicitsa bcr-abl peptide-specific CD4 immune response as measured by DTH invivo, by CD4+T cell proliferation ex vivo and by gamma interferonsecretion in an ELISPOT assay. However, no CD8 responses in HLA A0201patients and only weak responses in HLA A0301 patients were detectedusing a sensitive gamma interferon ELISPOT assay.

Wilms tumor protein I (WT1) is a zinc finger transcription factorexpressed during normal ontogenesis such as in fetal kidney, testis andovary. In adults, WTI expression is limited to low levels onhematopoietic stem cells, myoepithelial progenitor cells, renalpodocytes and some cells in testis and ovary. Recent demonstration thatWTI is over expressed in several types of leukemia suggested that WTIwould be an attractive target for immunotherapy; three peptide nonamersfrom WTI have been identified to generate a WTI specific cytotoxicresponse in the context of HLA 0201 and HLA 2402. However, as WTIprotein is a self-antigen, breaking tolerance is a potential concern.For stimulation of responses the strength of CD8 responses depends uponthe binding affinity of the target peptide to class I MHC molecules, thepeptide-HLA complex stability, and the avidity of the T cell receptorbinding for the peptide complex. Killing of native CML cells alsorequires adequate processing and presentation of the natural antigen.Therefore the lack of reproducible CD8 responses in these clinicaltrials could be the result of the biochemistry of these class Ipeptide-HLA interactions, which results in their weak immunogenicity tocytotoxic CDS cells, None of the native CML peptides reported to bind tohuman MHC bound the HLA pocket with high affinity. This may explain, inpart, the lack of a detectable immune response to bcr-abl peptides asproteins seen in patients with chronic myelogenous leukemia despite theappearance of this antigen in the CML cells.

In some antigenic systems peptide analogues are used to circumvent apoor immunogenic response. A high correlation has been found betweenoverall analogue peptide affinities for MHC class I molecules and invivo peptide immunogenicity in HLA-A2 Kb transgenic mice. A bettercorrelation with a peptide's ability to form stable HLA-A0201 complexesand immunogenicity has been reported. Improved immunogenicity inHLA-A020I/Kb transgenic mice also has been reported for analogues of aself-peptide, gp 100154-162, displaying both higher affinity and moreprolonged complex stability than the natural peptide.

To design peptide analogues several successful algorithms have beenutilized in which large protein sequences are scanned for the presenceof suitable binding motifs, leading to the identification of predictedantigens that have subsequently been experimentally validated. Analogsof antigenic peptides have been formulated by direct modifications ofMHC anchor positions, which are referred to as “MHC anchor-modifiedligands”, or modifications of TCR contact sites, which generally aretermed “altered peptide ligands”. The identification of peptide epitopeanalogues that strengthen the stability of the MHC-peptide complex invivo and in vitro is thought to enhance the potency of intrinsicallyweak immunogenic peptides for the activation and amplification ofrelevant T-cell subsets, This concept was originally described in amurine CD4⁺T cell model using HIV peptides (1), and now has beenextended to a variety of viral and tumor immunological systems.

Artificial variants of MHC class I-binding self-peptides have beendesigned (2). Since these variant peptides were foreign to the hostimmune system, a strong CTL response was induced. Unlike weak T cellresponses to self-peptide-MHC complexes, CTL responses to variantpeptides can be sustained for a longer period without causingannihilation of the clones due to insufficient signals for cell divisionor survival. Since a substantial fraction of such CTLs cross-react withnon-mutated self-peptides expressed in tumor cells in much smalleramounts, immunization with variant peptides may be a more efficientmethod to induce CTLs against tumors. The scoring system for MHC classI-binding peptides should provide a convenient method for design ofcross-reactive self-mimicking peptides for immunization.

The improved immunogenicity in vivo and relevance of MHC anchor-modifiedligands was first shown formally in human neoplastic disease in acontrolled study of patients with malignant melanoma using amelanoma-associated A0201 restricted peptide derived from gpl. It hasbeen shown recently with HLA-tetramer based detection methods that theparental Melan-A antigenic peptides are weak agonists which activateantigen-specific T cells suboptimally (3). In contrast, melan A peptideanalogues were identified that behaved as full agonists and induced fullT cell activation leading to strong tumor antigen-specific CTL responses(4).

Simple motifs and the statistical binding matrices can be used toperform a crude search for MHC-binding peptides. Unfortunately, thepresence of a simple sequence motif does not correlate well withbinding. Therefore these simple motifs are not always necessary orsufficient for binding. Only 30% of the peptides that carry such simplemotifs bind well when examined in a biochemical binding assay.Predictions of binding can be improved considerably when extended motifsare used, rather than the simple motifs. About 70% of the peptidescarrying an extended motif bind well.

Assuming that each amino acid in each position contributes a certainbinding energy independent of the neighboring residues and that thebinding of a given peptide is the result of combining the contributionsfrom the different residues, multiplying the relevant matrix valuesshould give an indication of the binding of the corresponding peptide.Such statistical matrix-driven predictions have been somewhat moresuccessful, thereby suggesting that MHC binding is to some extent theresult of a combinatorial specificity. The identification of analoguespeptides based on these methods has been applied recently to theidentification of CTL epitopes deduced from proteinase 3, melanomaantigen 3, mucin I and telomerase.

The weak immunogenicity of native bcr-abl fusion peptides, asdemonstrated by poor lysis of the cells, or the problem of toleranceusing native peptides from a self-antigen, such as WT1, has preventeduse of these native peptides as an effective vaccine against CML. A needexists in the art to develop therapeutic strategies using vaccinationagainst a truly tumor specific antigen that is also the oncogenicprotein requited for neoplasia. There is a need for improved syntheticpeptide analogues designed to elicit a greater immunogenic response. Theprior art is deficient in the lack of synthetic analogue peptides thatcould generate an immune response that not only recognizes theimmunizing epitopes, but that also cross reacts with the original nativepeptides. Specifically, the prior art is deficient in synthetic peptideanalogs with both improved HLA binding and improved ability to elicit agreater immunogenic response against cancer cells. The present inventionfulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

The present invention is directed to a synthetic peptide comprising asequence of amino acids containing at least a segment that is ananalogue of a native peptide that specifically binds to HLA A0201 or HLAA0301 molecules on a cell characteristic of a pathophysiologic state ina mammal. The synthetic peptide may be derived from native peptidescomprising a breakpoint region of the bcr-abl fusion protein or of theWT1 protein.

The present invention also is directed to a related synthetic peptidewith an amino acid sequence selected from YLKALQRPV (SEQ ID NO: 2),KQSSKALQV (SEQ ID NO: 4), KLSSKALQV (SEQ ID NO: 5), KLLQRPVAV (SEQ IDNO: 7), TLFKQSSKV (SEQ ID NO: 9), YLFKQSSKV (SEQ ID NO: 10), LLINKEEAL(SEQ ID NO: 12), LTINKVEAL (SEQ ID NO: 13), YLINKEEAL (SEQ ID NO: 14),YLINKEEAV (SEQ ID NO: 15), or YLINKVEAL (SEQ ID NO: 16), NMYQRNMTK (SEQID NO: 36), NMHQRVMTK (SEQ ID NO: 37), NMYQRVMTK (SEQ ID NO: 38),MYLGATLK (SEQ ID NO: 40), QMNLGVTLK (SEQ ID NO: 41), QMYLGVTLK. (SEQ IDNO: 42), FMYAYPGCNK (SEQ ID NO: 44), FMCAYPFCNK (SEQ ID NO: 45),FMYAYPFCNK (SEQ ID NO: 46), KLYHLQMHSR (SEQ ID NO: 48), KLSHLQMHSK (SEQID NO: 49), or KLYHLQMHSK (SEQ ID NO: 50).

The present invention also is directed to pharmaceutical compositioncomprising a therapeutically effective amount of the synthetic peptidesdescribed herein or a DNA encoding the synthetic peptide and a suitablecarrier.

The present invention is directed further to an immunogenic compositioncomprising an immunogenically effective amount of the synthetic peptidedescribed herein and a pharmaceutically acceptable carrier, adjuvant ordiluents or a combination thereof.

The present invention is directed further still to a method of treatinga cancer in a human. The pharmaceutical compositions described hereinare administered to the human. A heteroclitic response is induced bycytotoxic T-cells that recognize at least the analogue segment of thesynthetic peptides described herein against cancer cells presenting anative peptide from which the analogue segment is derived. Thus, thecytotoxic T-cells recognize or kill the cancer cells thereby treatingthe cancer. In a related method the present invention is directed to amethod of treating leukemia in a human using the pharmaceuticalcompositions comprising the synthetic peptide containing at least theanalogue segment derived from a native peptide of a WT1 protein or a DNAencoding the synthetic peptide.

The present invention is directed further still to a method of inducingformation and proliferation of human cytotoxic T cells that produce aheteroclitic immune response against cancer cells. Human immune cellsare contacted with a synthetic peptide containing at least the analoguesegment described herein. Thereby the formation and proliferation ofhuman cytotoxic T cells reactive against the activated cells presentingthe analogue segment of the synthetic peptide is induced. Theproliferating T cells will cross react with the cancer cells presentinga native peptide from which the analogue segment is derived such thatthe human cytotoxic T cells are thereby produce a heteroclitic immuneresponse against the cancer cells. The present invention also isdirected further to a related method of inducing formation andproliferation of human cytotoxic T cells that produce a heterocliticimmune response against leukemic cells in a human as described in themethod for inducing formation and proliferation of cytotoxic T cellsagainst a cancer cell.

The present invention is directed to a related method of inducing aheteroclitic response in a human. The immunogenic compositions describedherein are administered to a human to activate human immune cells. Theformation and proliferation of cytotoxic T cells against the activatedcells presenting the analogue segment of the synthetic peptide describedherein that comprises the immunogenic composition is induced thereby.The cytotoxic T cells will cross-react with a cancer cell presenting anative peptide from which the analogue segment is derived to induce theheteroclitic response The present invention is directed further to arelated method of inducing a heteroclitic response in a human againstleukemic cells as described in the method for inducing such heterocliticresponse against a cancer cell.

Other and further aspects, features, benefits, and advantages of thepresent invention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention are briefly summarized. Details of the above may be hadby reference to certain embodiments thereof which are illustrated in theappended drawings. These drawings form a part of the specification. Itis to be noted; however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

FIGS. 1A-1B show T2 stabilization assays using peptides derived fromb3a2 translocation (FIG. 1A) and b2a2 translocation (FIG. 1B).

FIGS. 2A-2B show the results of a CD8+ gamma interferon ELISPOT from ahealthy HLA A0201 donor (FIG. 2A) using p210C and p210F peptides andfrom a CML patient in chronic phase HLA A0201 (FIG. 2B) using the p210Cpeptide.

FIGS. 3A-3B show the results of a CD3+ gamma interferon ELISPOT from ahealthy HLA A0201 donor using the b2a2 A3-A5 peptides (FIG. 3A) and froma chronic myelogenous leukemia patient in chronic phase HLA A0201 (FIG.3B) using the b2a2 A3 peptide.

FIGS. 4A-4B show the results of cytotoxicity assays with T cells from ahealthy HLA A0201 donor using p2IOC, p21 OF and p!90B peptides (FIG. 4A)and the b2a2 A3 peptide (FIG. 4B).

FIGS. 5A-5E show binding of native and synthetic WT-1 peptides toHLAA0201 cells (FIG. 5A) and to HLA A0301 cells (FIGS. 5B-5E).

FIGS. 6A-6B show the results of a CD3⁺ gamma interferon ELISPOT (FIG.6A) and cytotoxicity (FIG. 6B) from a healthy HLA A0201 donor againstnative and synthetic peptide pulsed T2 cells.

FIGS. 7A-7D shows the results of a CD8⁺ (FIG. 7A) and CD3⁺ (FIGS. 7B-7D)gamma interferon ELISPOT from healthy HLA A0201 donors using native andsynthetic WT-1 peptides.

FIGS. 8A-8B show the results of cytotoxicity assays using CD8⁺T cellsstimulated with synthetic WT-1 AI peptides from a HLA A0201 donoragainst HLA matched CML blasts presenting native peptide sequences.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the present invention, there is provided asynthetic peptide comprising a sequence of amino acids containing atleast a segment that is analogue of a native peptide that specificallybinds HLA A0201 or HLA A0301 molecules on a cell characteristic of apathophysiologic state in a mammal. In this embodiment the analoguesegment may have a number of amino acids that is about 70% to about 130%of the number of amino acids in the native peptide. The number of aminoacids may be about 8 to about 12.

In all aspects of this invention the pathophysiologic state may be acancer.

The cancer may be a leukemia such as a chronic myelogenous leukemic.Alternatively, the cancer may breast cancer, lymphoma, mesothelioma,lung cancer, testicular cancer, or ovarian cancer. Additionally, in allaspects the mammal may be a human.

Furthermore, in all aspects of this embodiment the synthetic peptide maycomprise an immunogenic carrier linked thereto. Examples of carriers area protein, a peptide or an antigen-presenting cell. Representativeexamples of a protein or peptide are keyhole limpet hemocyanin, analbumin or a polyamino acid. A representative example of anantigen-presenting cell is a dendritic cell.

In one aspect of this embodiment the amino acids comprise a precursor tothe analogue segment which is a degradation product thereof. In thisaspect the precursor may be a bcr-abl fusion protein where the analoguesegment spans the breakpoint region of the fusion protein.Alternatively, the precursor may be WT1 where the analogue segmentreplaces a native peptide of WTL.

In a related aspect, the analogue segment is derived from a nativepeptide comprising a breakpoint region of a bcr-abl fusion protein. Thenative peptide may be a native p190-31a2 peptide. The native peptide maybe a native p210-b3a2 peptide and the amino acid sequence of theanalogue segment may be YLKALQRPV (SEQ ID NO: 2), KQSSKALQV (SEQ ID NO:4), KLSSKALQV (SEQ ID NO: 5), KLLQRPVAV (SEQ ID NO: 7), TLFKQSSKV (SEQID NO: 9), or YLFKQSSKV (SEQ ID NO: 10). Preferably, the amino acidsequence is YLKALQRPV (SEQ ID NO: 2), KLLQRPVAV (SEQ ID NO: 7) orYLFKQSSKV (SEQ ID NO: 10).

Alternatively, the native peptide may be a native p210-b2a2 peptide andthe amino acid sequence of the analogue segment may be LLINKEEAL (SEQ IDNO: 12), LTINKVEAL (SEQ ID NO: 13), YLINKEEAL (SEQ ID NO: 14), YLINKEEAV(SEQ ID NO: 15), and YLINKVEAL (SEQ ID NO: 16). Preferably the aminoacid sequence is YLINKEEAL (SEQ ID NO: 14).

In another aspect of this embodiment, the analogue segment is derivedfrom a native peptide comprising a WT1 protein. The amino acid sequenceof the WT1 derived analogue segment may be YMFPNAPYL (SEQ ID NO: 18),YLGEQQYSV (SEQ ID NO: 20), YLLPAVPSL (SEQ ID NO: 22), YLGATLKGV (SEQ IDNO: 24), YLNALLPAV (SEQ ID NO: 26), GLRRG1QDV (SEQ ID NO: 28), KLYFKLSHL(SEQ ID NO: 30), ALLLRTPYV (SEQ ID NO: 32), YMTWNQMNL (SEQ ID NO: 34),NMYQRNMTK (SEQ ID NO: 36), NMHQRVMTK (SEQ ID NO: 37), NMYQRVMTK (SEQ IDNO: 38), QMYLGATLK (SEQ ID NO: 40), QMNLGVTLK (SEQ ID NO: 41), QMYLGVTLK(SEQ ID NO: 42), FMYAYPGCNK (SEQ ID NO: 44), FMCAYPFCNK (SEQ ID NO: 45),FMYAYPFCNK (SEQ ID NO: 46), KLYHLQMHSR (SEQ ID NO: 48), KLSHLQMHSK (SEQID NO: 49), and KLYHLQMHSK (SEQ ID NO: 50).

In a related embodiment of this invention there is provided a syntheticpeptide with an amino acid sequence that may be one or more of YLKALQRPV(SEQ ID NO; 2), KQSSKALQV {SEQ ID NO: 4), KLSSKALQV (SEQ ID NO: 5),KLLQRPVAV (SEQ ID NO: 7), TLFKQSSKV (SEQ ID NO: 9), YLFKQSSKY (SEQ IDNO: 10), 5 LLINKEEAL (SEQ ID NO: 12), LTINKVEAL (SEQ ID NO: 13),YLINKEEAL (SEQ ID NO: 14), YLINKEEAV (SEQ ID NO: 15), or YLINKVEAL (SEQID NO: 16). Alternatively, a synthetic peptide is provided with an aminoacid sequence that may be one or more of NMYQRNMTK (SEQ ID NO: 36),NMHQRVMTK (SEQ ID NO: 37), NMYQRVMTK (SEQ ID NO: 38), QMYLGATLK (SEQ IDNO: 40), QMNLGVTLK10 (SEQ ID NO: 41), QMYLGVTLK (SEQ ID NO: 42),FMYAYPGCNK (SEQ ID NO: 44), FMCAYPFCNK {SEQ ID NO: 45), FMYAYPFCNK (SEQID NO: 46), KLYHLQMHSR (SEQ ID NO: 48), KLSHLQMHSK (SEQ ID NO: 49), orKLYHLQMHSK (SEQ ID NO: 50).

In another related embodiment there is provided a pharmaceuticalcomposition comprising a therapeutically effective amount of thesynthetic peptide of claim 1 or a DNA encoding the synthetic peptide;and a pharmaceutically acceptable carrier. In aspects of this embodimentwhere the pharmaceutical composition comprises a DNA encoding thesynthetic peptide, the DNA may be inserted into a vector or into anantigen-presenting cell. An example of an antigen presenting cell is adendritic cell.

In one aspect of this embodiment an analogue segment comprising thesynthetic peptide is derived from a native p210-b.3a2 peptide, a nativep210-b2a2 peptide or a native p190-ela2 peptide. The amino acidsequences for these analogue segments are as identified supra forp210˜b3a2 and p210-b2a2 derived analogues. In a related aspect of thisembodiment an analogue segment comprising the synthetic peptide isderived from a native peptide comprising the WT-1 protein. These aminoacid sequences for these WTI-derived analogue segments are as identifiedsupra for WT-1 derived analogues.

In still another related embodiment of the present invention, there isprovided an immunogenic composition comprising an immunogenicallyeffective amount of the synthetic peptides described supra and apharmaceutically acceptable carrier, adjuvant or diluent or acombination thereof. The carrier may be a protein, a peptide or anantigen-presenting cell linked to the synthetic peptide Examples of aprotein or peptide carrier are keyhole limpet hemocyanin, an albumin ora polyamino acid. An example of an antigen-presenting cell is adendritic cell. The synthetic peptides and analogue segments comprisingthe synthetic peptides are as described supra. In aspects of thisembodiment, the analogue segments may be derived from those nativepeptides and the amino acid sequences may be those sequences describedsupra for a pharmaceutical composition. In another embodiment of thepresent invention, there is provided a method of inducing formation andproliferation of hum an cytotoxic T cells that produce a heterocliticimmune response against cancer cells, comprising contacting human immunecells with the synthetic peptides described supra to activate the immunecells; and inducing formation and proliferation of human cytotoxic Tcells reactive against the activated cells presenting at least theanalogue segment of the synthetic peptide, where the proliferating Tcells will cross react with the cancer cells presenting a native peptidefrom which said analogue segment is derived such that the humancytotoxic T cells are capable of producing a heteroclitic immuneresponse against the cancer cells.

In this embodiment the method further comprises providing a DNA encodingthe synthetic peptide and expressing the DNA. The DNA may be insertedinto a suitable vector. Alternatively, the DNA may be inserted into anantigen-presenting cell. An example of an antigen-presenting cell is adendritic cell. In one aspect of this embodiment the human immune cellsare contacted in vivo in an individual having a cancer. In a relatedaspect the human immune cells are contacted in vivo in a donor and themethod further comprises obtaining the cytotoxic T cells from the donorand infusing the cytotoxic T cells into a recipient having a cancer.

In another aspect the cells are contacted ex vivo and the method furthercomprises obtaining the human immune cells from a donor prior to thecontacting the human immune cells with the pharmaceutical compositionand infusing the activated immune cells into an individual having acancer prior to the inducing formation and proliferation of cytotoxic Tcells. In a related aspect the cells are contacted ex vivo and themethod further comprises obtaining human immune cells from a donor priorto contacting the human immune cells. In this aspect, both contactingthe human immune cells and formation and proliferation of the cytotoxicT-cells occurs ex vivo, infusing the cytotoxic T-cells into anindividual having a cancer.

In all aspects of this embodiment, representative human immune cells maybe peripheral blood mononuclear cells, bone marrow cells, dendriticcells, or macrophages. The synthetic peptides and analogue segmentscomprising the synthetic peptides are as described supra. In aspects ofthis embodiment the analogue segments comprising the synthetic peptidemay be derived from a native p210˜b3a2 peptide, a native p210-b2a2peptide, a native p190-ela2 peptide or from a native WT-1 peptide asdescribed supra, Furthermore, the amino acid sequences of thesesynthetic peptides or the analogue segments comprising the syntheticpeptides may have an amino acid sequence as identified supra forp210-b3a2-, p210-b2a2- or WT-1-derived analogues.

In a related aspect WT-1 analogue segments and p210-b3a2 and p210-b2a2derived analogue segments may induce a heteroclitic response againstSeukemic cells. Representative leukemic cells are chronic myelogenousleukemic cells. In another related aspect WT-1 analogue segments mayinduce a heteroclitic response against from breast cancer, lymphoma,mesothelioma, lung cancer, testicular cancer, or ovarian cancer.

In yet another embodiment of the present invention there is provided amethod of treating a cancer in a human, comprising administering thepharmaceutical compositions described herein to the human; and inducinga heteroclitic response by cytotoxic T-cells that recognize at least theanalogue segment of said synthetic peptide against cancer cellspresenting a native peptide from which said analogue segment is derived,said cytotoxic T cells recognizing or killing said cancer cells therebytreating the cancer.

The synthetic peptides or DNAs encoding the synthetic peptides are asdescribed supra. In aspects of this embodiment the analogue segmentcomprising the synthetic peptides may be derived from a native p190 ela2peptide, a native p210-b3a2 peptide, a native p210˜b2a2 peptide, or froma native WT-1 peptide as described supra. Furthermore, the amino acidsequences of these synthetic peptides or the analogue segmentscomprising the synthetic peptides are as identified supra forp210-b3a2-, p210-b2a2- or WT-1-derived analogue segments.

In one aspect WT-1 at least the analogue segments comprising thesynthetic peptides and p210-b3a2 and p210-b2a2 derived syntheticpeptides may treat a leukemia. A representative leukemia is chronicmyelogenous leukemia. In another aspect WT-1 at least the analoguesegments comprising the synthetic peptides may treat breast cancer,lymphoma, mesothelioma, lung cancer, testicular cancer, or ovariancancer.

In a related embodiment, there is provided a method of inducing aheteroclitic immune response in a human, comprising administering to thehuman an effective amount of the immunogenic compositions describedsupra; activating human immune cells with the immunogenic composition;and inducing formation and proliferation of human cytotoxic T cellsagainst the activated cells presenting at least the analogue segment ofthe synthetic peptide comprising the immunogenic composition. In thismethod, the human cytotoxic T cells will cross-react with a cellcomprising a cancer presenting a native peptide from which said analoguesegment is derived, thereby inducing the heteroclitic immune response.

In one aspect of this embodiment, the human may have an active cancer,may be in remission from cancer or may be at risk of developing acancer. In an alternative aspect of this embodiment human donates thecytotoxic T-cells to an individual having an active cancer, is inremission from cancer or is at risk of developing a cancer.

In aspects of this embodiment, the immunogenic compositions, the humanimmune cells and the synthetic peptides and the analogue segmentscomprising the synthetic peptides are as described supra. In one aspectat least WT-1 analogue segments and p210-b3a2 and p210-b2a2 derivedanalogue segment comprising synthetic peptides may induce a heterocliticresponse in the human against leukemia.

A representative leukemia is chronic myelogenous leukemia. In anotheraspect at least WT-1 analogue segments may induce a heterocliticresponse in the human against breast cancer, lymphoma, mesothelioma,lung cancer, testicular cancer, or ovarian cancer.

Provided herein are synthetic immunogenic peptides with an amino acidsequence containing at least an analogue segment of a native peptidethat demonstrates improved binding over the native peptides to HLA A0201or to HLA A0301 complexes. These synthetic peptides or analogue segmentscan stimulate T-cells to cross-react with the native peptides thuseliciting a heteroclitic immune response that will recognize or killcells presenting the native peptides. Such cells are characteristic of apathophysiological state, for example, but not limited to, a cancer. Atleast the analogue segments comprising the synthetic peptides will bindwith more affinity to the HLA class I and class II molecules that areinstrumental in presenting the analogue segments to the T-cells than thenative peptide itself.

The synthetic peptide analogue segments are designed by making one ortwo amino acid substitutions in anchor or auxiliary residues. Althoughthe native peptides particularly described herein are nonamersencompassing the anchor or auxiliary residues, analogues may be designedhaving about 70% to about 130% of the amino acids in the native peptide.In the instant invention the synthetic peptide analogues may have about8-12 amino acids. Such substitutions are determined by a bioinformaticmodel system (BIMAS) which uses a matrix approach to predict binding andranks the peptides based on predicted binding to the HLA molecule. Theamino acid sequences and predicted score for binding to HLA A0201 andHLA A0301 are generated by online software BIMAS available athttp://bimas.dcrt.nih.gov/cgi-bin/molbio/ken parker comboform andSYFPEITHI available at http://syfpeimi-bmi-heidelberg.com/. Thesynthetic peptide may be a precursor to the analogue segment which maybe a degradation product of the synthetic peptide. Such precursor may bea bcr-able fusion protein such that the synthetic analogue spans thebreakpoint region of the fusion protein. Alternatively, the precursormay be a WT1 protein such that the analogue segments replace a nativepeptide sequence within WT1.

Additionally, the synthetic peptide is or comprises analogue segmentsthat may be analogues of the breakpoint region of the bcr-abl fusionprotein, which is the oncogenic protein required for neoplasia inchronic myelogenous leukemia. The synthetic peptides are or compriseanalogue segments derived from the junctional sequences of p210-b3a2,p210-b2a2 and p19Q-ela2. More preferably, the synthetic peptides or theanalogue segments are derived from p210-b3a2 and p210-b2a2 in whichsingle or double amino acid substitutions were introduced into thepeptides at key HLA A0201 binding positions. These high affinity peptideanalogues were able to generate specific CD8+T cells far moreefficiently than the native peptides and are capable of stimulatinghuman CD8+ CTL heteroclitic responses that cross-react with the nativesequences presented on leukemic cells.

Preferred synthetic peptides or analogue segments are the p210-b.3a2analogues p210C, p210D, p210E, and p210F more preferably p210C, and thep210-b2a2 analogues b2a2 A3, b2a2 A4 and b2a2 A5, more preferably b2a2A3, Table I shows the amino acid sequences and binding predictions ofnative and synthetic analogues. The underlined K in the b3a2 andunderlined E in b2a2 are the amino acids in the breakpoint. Boldface,italic residues represent modification from the native sequence.

TABLE 1 “Native Analogue BIMAS Name sequence sequence score CMLA2SSKALQRPV 0.003 SEQ ID NO: 1 p210F

KALQRPV 2240 SEQ ID NO: 2 CMLA3 KQSSKALQR 0.005 SEQ ID NO: 3 p210AKQSSKALQ

24.681 SEQ ID NO: 4 p210B K

SSKALQ

243.432 SEQ ID NO: 5 p210Cn KALQRPVAS 0.013 SEQ ID NO: 6 p210C K

LQRPVA

900.689 SEQ ID NO: 7 p210Dn TGFKQSSKA 0.120 SEQ ID NO: 8 p210D T

FKQSSK

257.342 SEQ ID NO: 9 p210E

FKQSSK

1183.775 SEQ ID NO: 10 b3a2A LTINKEEAL 0.247 SEQ ID NO: 11 b3a2 A1 L

INKEEAL 17.795 SEQ ID NO: 12 b3a2 A2 LTINK

EAL 21.996 SEQ ID NO: 13 b3a2 A3

INKEEAL 48.151 SEQ ID NO: 14 b3a2 A4

INKEEA

156.770 SEQ ID NO: 15 b3a2 A5

INK

EAL 110.747 SEQ ID NO: 16

The synthetic immunogenic peptides may be analogue segments or compriseanalogue segments derived from WT1 protein. Computer predictionanalysis, as described herein, predicted synthetic peptides analoguesderived from nonamer sequences of the WT1 protein in which singleamino-acid substitutions were introduced at HLA A0201 binding and singleor double amino acid substitutions were introduced at A0301 bindingpositions. These synthetic peptide analogues or analogue segments wereable to generate specific CD8⁺ or CD3⁺T cells far more efficiently thanthe native peptides and are capable of stimulating human CD8⁺ or CD3⁺CTLheteroclitic responses that cross-react with the native sequencespresented on leukemic cells or on other cells that present these nativeWT-1 peptides. Tables 2 and 3 show the amino acid sequences and bindingpredictions of native WT-1 and synthetic WT-1 peptide analogues.Boldface, italic residues represent modifications from the nativesequence.

TABLE 2 HLA 0201 native peptides from WT-1 and synthetic analoguesNative Analogue BIMAS Name sequence sequence score WT-1 A RMFPNAPYL 313SEQ ID NO: 17 WT-1 A1

MFPNAPYL 1444 SEQ ID NO: 18 WT-1 B SLGEQQYSV 285 SEQ ID NO: 19 WT-1 B1

LGEQQYSV 1311 SEQ ID NO: 20 WT-1 C ALLPAVPSL 181 SEQ ID NO: 21 WT-1 C1

LLPAVPSL %36 SEQ ID NO: 22 WT-1 D NLGATLKGV 159 SEQ ID NO: 23 WT-1 D1

LGATLKGV 735 SEQ ID NO: 24 WT-1 E DLNALLPAV 11 SEQ ID NO: 25 WT-1 E1

LNALLPAV 735 SEQ ID NO: 26 WT-1 F GVFRGIQDV 51 SEQ ID NO: 21 WT-1 F1 G

RGIQDV 591 SEQ ID NO: 28 WT-1 G KRYFKLSHL 1 SEQ ID NO: 29 WT-1 G1 K

YFKLSHL 550 SEQ ID NO: 30 WT-1 H ALLLRTPYS 1 SEQ ID NO: 31 WT-1 H1ALLLRTPY

1415 SEQ ID NO: 32 WT-1 J CMTWNQMNL 15 SEQ ID NO: 33 WT-1 J1

MTWNQMNL 70 SEQ ID NO: 34

TABLE 3 HLA 0201 native peptides from WT-1 and synthetic analoguesNative Analogue BIMAS Name sequence sequence score A3 WT-1 A NMHQRNMTK40 SEQ ID NO: 35 A3 WT-1 A1 NM

QRNMTK 200 SEQ ID NO: 36 A3 WT-1 A2 NMHQR

MTK 120 SEQ ID NO: 37 A3 WT-1 A3 NM

QR

MTK 600 SEQ ID NO: 38 A3 WT-1 B QMNLGATLK 20 SEQ ID NO: 39 A3 WT-1 B1 QM

LGATLK 100 SEQ ID NO: 40 A3 WT-1 B2 QMNLG

TLK 60 SEQ ID NO: 41 A3 WT-1 B3 QM

LG

TLK 300 SEQ ID NO: 42 A3 WT-1 C FMCAYPGCNK 30 SEQ ID NO: 43 A3 WT-1 C1FM

AYPGCNK 150 SEQ ID NO: 44 A3 WT-1 C2 FMCAYP

CNK 90 SEQ ID NO: 45 A3 WT-1 C1 FM

AYP

CNK 450 SEQ ID NO: 46 A3 WT-1 D KLSHLQMHSR 18 SEQ ID NO: 47 A3 WT-1 D1KL

HLQMHSR 90 SEQ ID NO: 48 A3 WT-1 D2 KLSHLQMHS

90 SEQ ID NO: 49 A3 WT-1 D3 KL

HLQMHS

450 SEQ ID NO: 50

The present invention also provides a pharmaceutical composition of atherapeutic amount of the synthetic peptides or analogue segments or agenetic sequence or DNA encoding the same and a pharmaceutical carrier,as is known in the art. The pharmaceutical composition may be formulatedwith the pharmaceutical carrier for administration by any of the manytechniques known to those of skill in the art. For example, thepharmaceutical composition may be administered parenterally,intravenously, subcutaneously, intradermally, intramucosally, topically,orally, or by inhalation.

Therefore, it is contemplated that the synthetic peptides or analoguesegments or pharmaceutical compositions thereof may be used in thepreparation of an immunogenic composition suitable to effectimmunization of a subject. The immunogenic composition may comprise acarrier or a suitable adjuvant to boost immune response or a combinationthereof, as are known in the art. The immunogenic composition furthermay comprise a diluent standard in the art as described herein. Theimmunogenic composition may comprise a vaccine.

A carrier may comprise one or more proteins or peptides. Examples ofcarriers are well known and may be, although not limited to keyholelimpet hemocyanin, an albumin, such as human serum albumin or apolyamino acid. Additionally, a carrier may comprise a liveantigen-presenting cell, such as a dendritic cell, which presents thesynthetic peptides described herein. A suitable adjuvant may be Freund'sadjuvant, aluminum phosphate, aluminum hydroxide, alum, QS21, BCG. Thesecompositions further may comprise a physiologically acceptable diluent,e.g., water, phosphate buffered saline or saline. Additionally, agenetic sequence encoding a synthetic peptide or an analogue segmentthereof may be delivered as naked DNA to an individual via appropriatemethods known in the art. Alternatively, the genetic sequence may beintroduced or inserted into a suitable vector, such as for example, butnot limited to, attenuated viral or bacterial vectors, as are standardin the art. Furthermore, the naked DNA or vectors comprising the geneticsequence or DNA may be transduced into an antigen-presenting cell, e.g.,a dendritic cell. The genetic sequence, DNA, vector or transducedantigen-presenting cell may be introduced into an individual in need ofthe treatment or into a healthy donor whereupon the DNA encoding thegenetic sequence expresses the synthetic peptide to elicit a cytotoxicT-cell response. Donor T-cells may then be infused into a patient inneed thereof.

The pharmaceutical or immunogenic compositions may be used to treat adisease or a condition such as cancer. Administration of the syntheticpeptides or analogue segments comprising the pharmaceutical compositionsinduces a heteroclitic response against native peptides expressed on thecancer cells thereby effecting a therapeutic result. Native peptides ofthe breakpoint region of bcr-abl proteins and native peptides of WT-1protein are expressed on leukemic cells in chronic myelogenous leukemia.Native WT-1 peptides are expressed on other leukemic cells and,additionally, on cancerous cells of different solid tumors. Such cancersmay be, although not limited to, breast, lymphoma, mesothelioma, lung,testicular, or ovarian cancers.

It is contemplated that the synthetic peptides or synthetic analoguesegments thereof or genetic sequences encoding the same or thepharmaceutical or the immunogenic compositions thereof can induce humancytotoxic T cells to produce a heteroclitic immune response againstcancerous cells, for example, leukemic cells. Contacting human immunecells with at least the analogue segment that is or comprises thesynthetic peptides activates the immune cells to induce formation andproliferation of human cytotoxic T cells that will recognize or reactagainst a cell presenting the synthetic peptide. Such cytotoxic T cellscross react with human cells presenting the native peptides from whichthe analogue segment is derived thereby producing a heterocliticresponse.

One of ordinary skill in this art would recognize the word “contacting”in terms of activating target immune cells to elicit a subsequent immuneresponse as referring to any suitable delivery method of bringing animmunogenic agent into contact with the target cells. In vitro or exvivo this is achieved by exposing the target cells to the agent in asuitable medium. For in vivo applications, any known method ofadministration is suitable as described herein.

Thus, the synthetic peptides or analogue segments thereof describedherein may be used to activate T-cells ex vivo or in vivo. In vivo, thesynthetic peptides or analogue segments thereof or DNA encoding the samemay be administered to a patient or to a healthy donor to inducecytotoxic T-cells If administered to a donor these cytotoxic T-cells areobtained from the donor and infused into an individual in need of them,such as an individual with an active cancer, in remission from a canceror at risk for developing a cancer.

Ex vivo, the T cells are obtained from a patient or from a healthy donorand are incubated in the presence of antigen presenting cells and asynthetic peptide or at least an analogue segment thereof to activatethe T-cells. The activated T-cells subsequently are infused back intothe patient where they will recognize and/or destroy cells presentingthe native peptide. Alternatively, human immune cells may be incubatedwith the synthetic peptide or at least an analogue segment thereofwhereupon the activated immune cells are infused back into the patientto induce T-cell production against both the activated cells and cellpresenting the native peptide. Examples of immune cells may beperipheral blood mononuclear monocytic cells, bone marrow cells,dendritic cells, or macrophages.

It is contemplated further that administration of the synthetic peptideor at least an analogue segment thereof or pharmaceutical compositionsthereof induces an immune response in a subject, preferably, althoughnot limited to, a CD8/HLA A or CD3/HLA A class I immune response. Assuch, the synthetic peptides or at least an analogue segment thereof maybe used in a method of immunizing a subject against a pathophysiologiccondition or disease presenting HLA molecules, e.g., a leukemia, such aschronic myelogenous leukemia- Additionally, WT-1 synthetic peptides orat least analogue segments thereof may be used to induce an immuneresponse in a subject with other leukemias or cancers such as, althoughnot limited to, breast, lymphoma, mesothelioma, lung, testicular, orovarian cancers. As used herein, immunizing or immunization of a subjectencompasses full and partial immunization whereby the subject becomesfully immune to the condition or partially immune to the condition. Thesubject may be a mammal, preferably a human. The subject may have acondition or disease which may be active or in remission, prior toimmunization. Alternatively, if at risk for developing the disease orcondition, the subject may be immunized prior to such development. Oneof ordinary skill in the art would be able to assess the risk factors,such as environmental risk factors or personal risk factors, such asfamily history, genetic makeup or behavior, to make a determination ofrisk in the subject.

The pharmaceutical compositions and immunogenic compositions may beadministered one or more times to achieve a therapeutic or animmunogenic effect. It is well within the skill of an artisan todetermine dosage or whether a suitable dosage comprises a singleadministered dose or multiple administered doses. An appropriate dosagedepends on the subject's health, the progression or remission of thedisease, the route of administration and the formulation used.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

Example 1 Synthetic Peptides

Each of the peptides utilized in this study was purchased and wassynthesized by Genemed Synthesis Inc, CA using fluorenylmethoxycarbonylchemistry, solid phase synthesis and purified by high pressure liquidchromatography. The quality of the peptides was assessed byhigh-performance liquid chromatography analysis and the expectedmolecular weight was observed using matrix-assisted laser desorptionmass spectrometry. Peptides were sterile and >90% pure. The peptideswere dissolved in DMSO and diluted in phosphate-buffered saline (PBS) atpH 7.4 or saline to give a concentration of 5 mg/ml and were stored at−80′C. For in vitro experiments an irrelevant control peptide, HLA A24consensus, was used.

Example 2 Cell Lines

Cell lines were cultured in RPMI 1640 medium supplemented with 5% FCS,penicillin, streptomycin, 2 mM glutamine and 2-mercaptoethanol at 37° C.in humidifier air containing 5% CO₂. SKLY-16 is a human B cell lymphomaexpressing HLA A0201 and T2 is a human cell line lacking TAP1 and TAP2and therefore unable to present peptides derived from cytosolicproteins.

Example 3 T2 Assay for Peptide Binding and Stabilization of HLA A0201Molecules

T2 cells (TAP⁻, HLA-A0201+) were incubated overnight at 27° C. at 1×10E6 cells/ml in FCS-free RPMI medium supplemented with 5 μg/ml human β2m(Sigma, St Louis, Mo.) in the absence, i.e., negative control, orpresence of either a positive reference tyrosinase peptide or testpeptides at various final concentrations of 50, 10, 1, and 0.1 fig/mlFollowing a 4 h incubation with 5 μg/ml brefeldin A (Sigma), T2 cellswere labeled for 30 min at 4° C. with a saturating concentration ofanti-HLA-A2.1 (BB7.2) mAb, then washed twice. The cells then wereincubated for 30 min at 4° C. with a saturating concentration ofFITC-conjugated goat IgG F(ab′)₂ anti-mouse Ig (Caltag, South SanFrancisco, Calif.), washed twice, fixed in PBS/1% paraformaldehyde andanalyzed using a FACS Calibur cytofluorometer (Becton Dickinson,Immunocytometry systems, San Jose, Calif.).

The mean intensity of fluorescence (MIF) observed for each peptideconcentration, after subtraction of the MIF observed without peptide,was used as an estimate of peptide binding. Stabilization assays wereperformed similarly. Following initial evaluation of peptide binding attime 0, cells were washed in RPMI complete medium to remove freepeptides and incubated in the continuous presence of 0.5 μg/mlbrefeldin-A for 2, 4, 6, and 8 hours. The amount of stablepeptide-HLA-A2.1 complexes was estimated as described above by indirectimmunofluorescence analysis. The half time of complexes is an estimateof the time required for a 50% reduction of the time mean intensity offluorescence value.

Example 4 Competition Radioimmunoassay

Target cells were washed two times in PBS with 1% bovine serum albumin(Fisher Chemicals, Fairlawn, N.J.). Cells were resuspended at 10⁷/ml onice and the native cell surface bound peptides were stripped for 2minutes at 0° C. using citrate-phosphate buffer in the presence of beta₂microglobulin 3 mg/ml. The pellet was resuspended at 5 to 10×10⁶cells/ml in PBS/1% BSA in the presence of 3 mg/ml beta₂ microglobulinand 30 mg/ml deoxyribonuclease and 200 ml aliquots were incubated withHLA-specific peptides for 10 min at 20° C.

Binding of ¹²⁵I-labeled peptide with or without competitive unlabeledpeptide was done for 30 min at 20° C. Total bound ¹²⁵I was determinedafter two washes using PBS/2% BSA and one wash with PBS. Relativeaffinities were determined by comparison of escalating concentrations ofthe test peptide versus a known binding peptide. Peptides ofaffinities<500 nM were chosen for use.

A specificity analysis of the binding of peptide to HLA on the live cellsurface (SKLY-16) was conducted to confirm that the binding was to theappropriate HLA molecule and to characterize its restriction. Thisincluded competition with excess unlabeled peptides known to bind to thesame or disparate HLA molecules and use of target cells which expressedthe same or disparate HLA types. This assay was performed on live freshor 0.25% paraformaldehyde-fixed human peripheral blood mononuclear cells(PBMC), leukemia cell lines and EBV-transformed T-cell lines of specificHLA types. The relative avidity of the peptides found to bind MHCmolecules on the specific cells were assayed by competition assays asdescribed above against ¹²⁵I-labeled peptides of known high affinity forthe relevant HLA molecule, e.g., tyrosinase or HBV peptide sequence.

Example 5 In Vitro Immunization and Human T Cell Cultures

After informed consent, peripheral blood mononuclear cells fromHLA-A0201 positive healthy donors and chronic myeloid leukemia patientswere obtained by Ficoll-density centrifugation. Peripheral blooddendritic cells (DCs) were generated as follows: Monocyte-enrichedperipheral blood mononuclear cell fractions were isolated, using aplastic adherence technique, from total peripheral blood mononuclearcells. The plastic-adherent cells were cultured further in RPMI 1640medium supplemented with 1-5% autologous plasma, 1000 U/mL recombinanthuman interleukin (IL)-4 (Schering-Plough, N.J.), and 1000 U/mLrecombinant human granulocyte-macrophage colony-stimulating factor(GM-CSF) (Immunex, Seattle).

On days 2 and 4 of incubation, part of the medium was exchanged forfresh culture medium supplemented with IL-4 and GM-CSF and culture wascontinued. On day 6, half of the medium was exchanged for culture mediumsupplemented with IL-4, GM-CSF, and 10 ng/mL recombinant human tumornecrosis factor (TNF)-alpha (R&D 5 system) and 500 ng/ml of trimericsoluble CD40L (Immunex, Seattle). On day 9, the cells were harvested andused as monocyte-derived dendritic cells for antigen stimulation. Thecells generated expressed dendritic cell-associated antigens, such asCD80, CD83, CD86, and HLA class 1 and class II on their cell surfaces(data not shown).

T lymphocytes were isolated from the same donors by use of negativeselection by depletion with an anti-CD11b, anti-CD56 and CD19 monoclonalantibody (Miltenyi, Calif.) A total of 1×10 E6 of pure T lymphocyteswere cultured with 1×10 E5 autologous dendritic cells in RPMI1640 mediumsupplemented with 5% heat-inactivated human autologous plasma withbcr-abl synthetic peptides at a concentration of 10 μg/mL and b2microglobulin at 2 μg/ml in 24 well plates in the presence of 5-10 ng/mLrecombinant human IL-7 (Genzyme) and 0.1 ng/ml of IL-12.

After culture for 3 days 20 U/ml of IL-2 was added. After 10 days, 1×10E6 cells were stimulated again by adding 2×10 E5 autologous magneticallyisolated CD 14⁺ monocytes together with 10 ng/ml of IL-7 and 20 U/ml ofIL-2 and peptide at a concentration of 10 μg/mL. In some cases, afterculture for another 7 days, the cells were stimulated a third time inthe same manner. After the second or third stimulation, CD8 T cells wereisolated magnetically and cytotoxicity and gamma-IFN secretion of thesecells were examined.

Example 6 Gamma Interferon ELISPOT

HA-Multiscreen plates (Millipore, Burlington, Mass.) were coated with100 μl of mouse-anti-human IFN-gamma antibody (10 μg/ml; clone 1-D1K,Mabtech, Sweden) in PBS, incubated overnight at 4° C., washed with PBSto remove unbound antibody and blocked with RPMI/autologous plasma for 1h at 37° C. Purified CD8⁺T cells, more than 95% pure, were plated at aconcentration of 1×10⁵/well. T cells were stimulated with 1×10 E4 T2cells per well, pulsed with 10 μg/ml of B2-microglobulin (Sigma, St.Louis) and either 50 μg/ml of test peptide, positive control influenzamatrix peptide, or irrelevant control peptide at a final volume of100-200 μl/well. Control wells contained T2 cells with or without CD8⁺cells. Additional controls included medium or CD8⁺ alone plus PBS/5%DMSO diluted according to the concentrations of peptides used forpulsing T2 cells.

After incubation for 20 hours at 37° C., plates were washed extensivelywith PBS/0.05% Tween and 100 μl/well biotinylated detection antibodyagainst human IFN-g at 2 μg/ml (clone 7-B6-1, Mabtech, Sweden) wereadded. Plates were incubated for an additional 2 hours at 37° C. andspot development was performed. Spot numbers were automaticallydetermined with the use of a computer-assisted video image analyzer withKS EL1SPOT 4.0 software (Carl Zeiss Vision, Germany).

Example 7 Cytotoxicity Assay

The presence of specific CTLs was measured in a standard 4 h-chromiumrelease assay. 4×10 E6 targets were labeled with 300 μCi of Na₂ ⁵¹Cr0₄(NEN Life Science Products, Inc. Boston, Mass.) for 1 hour at 37° C.After washing, cells at 2×10 E6/ml were incubated with or withoutsynthetic peptides at a concentration of 10 μg/ml for 2 hours at 20° C.in presence of B₂ microglobulin at 3 μg/ml. After washing bycentrifugation, target cells were resuspended in complete media at 5×10E4 cells per ml and plated in a 96 well U-bottom plate (BeetonDickinson®, NY) at 5×10 E3 cells per well with effector cells ateffector to target ratios (E/T) ranging from 100:1 to 10:1. Plates wereincubated for 5 hours at 37° C. in 5% CO₂.

Supernatant fluids were harvested and radioactivity was measured in agamma counter. Percent specific lysis was determined from the followingformula: 100×[(experimental release minus spontaneous release)/(maximumrelease minus spontaneous release)]. Maximum release was determined bylysis of targets in 2.5% Triton X-100.

Example 8 Identification and Generation of Peptides with a HighProbability to Bind to HLA 0201

Amino acid sequences of the human b3a2 and b2a2 fusion proteins werescanned for peptides with potential binding capacity for HLA A0201, asubtype encompassing 95% of the HLA-A02 allele. HLA-A0201 is expressedin about 40% of the Caucasian population. No peptides with high orintermediate affinity were identified in the native b3a2 or b2a2 fusionproteins with > than 1 minute of predicted half life. One peptide thatdoes not exhibit the consensus HLA 0201 binding motifs has beendescribed but it has weak avidity to MHC.

Based on this information and by using the software of theBioinformatics & Molecular Analysis Section (National Institutes ofHealth, Washington, D.C.) available at http://bimas.dcrtnihgov/cgi-bin/molbio/ken parker comboform This software ranks 9-mer or10-mer peptides on a predicted half-time dissociation coefficient fromHLA class I molecules (Pinilla, et al Curr Opin Immunol, 11(2): p.193-202 (1999)). Analogue peptides were designed by changing one or bothanchor amino acids or additional amino acids adjacent to anchor aminoacids. Single or double amino acid substitutions were introduced at HLAA0201 preferred residues at positions 1, 2, 6 and 9 {Table 1) to yieldsequences that had comparatively high binding scores predicted for HLAA0201 molecules.

The predicted half life for binding to HLA A0201 was greater than 240minutes in four synthetic peptides and less than 240 in seven. All thenative peptides were predicted to have less than an hour of half life.Most of the substitutions affected the primary or secondary anchormotifs, i.e., leucine in position 2 or valine in position 9 or position6, but in some cases, a tyrosine was substituted in position 1. Thissubstitution has been shown to stabilize the binding of position 2anchor residue.

Example 9

Binding of HLA-A0201 by synthetic peptides analogues of b2a2 and b3a2native peptides. The immunogenicity of MHC class I-restricted peptidesrequires the capacity to bind and stabilize MHC class I molecules on thelive cell surface. Moreover the computer prediction models above haveonly 60-80% predictive accuracy. Direct measurement of the strength ofthe interaction between the peptides and the HLA-A0201 molecule was madeusing a conventional binding and stabilization assay that uses theantigen-transporting deficient (TAP2-) HLA-A0201 human T2 cells. T2cells lack TAP function and consequently are defective in properlyloading class I molecules with antigenic peptides generated in thecytosol. The association of exogenously added peptides withthermolabile, empty HLA-A2 molecules stabilizes them and results in anincrease in the level of surface HLA-A0201 recognizable by specific mAbsuch as BB7.2.

Seven out of the eleven peptides designed to have higher binding scoresexhibited a relatively high binding affinity for HLA A0201 molecules asmeasured by the T2 assay (FIGS. 1A-1B). A rough correlation betweenbinding scores and binding affinity was established, thus indicating thepartial utility of the computer generated binding scores for predictingpeptides that will bind to MHC class I molecules on live cells. Some ofthese peptides demonstrated the same order of binding affinity as thatof viral antigen such as influenza, which are among the most potentknown antigens for CTL induction. In four cases a good correlationbetween computer predicted half-life and T2 stabilization was not found.

One of the peptides derived from b3a2, p210C, was mutated from a peptidethat did not have a good prediction score. However, the native sequenceis able to bind HLA A0201 weakly and at the same level that thepreviously described CMLA2 peptide. To design p210C, a neutral alaninein position two was substituted by a leucine and a serine in positionnine was substituted by a valine. p210C has a high BIMAS score thatcorrelated with T2 binding assay data (FIG. 1A).

p210F is a peptide derived from the sequence previously described byYotonda et al, CM.LA2, shown to be a weak binder in the T2 assay. Inp210F the two serines in position one and two were substituted by a tyrosine and a leucine. The BIMAS prediction showed a 700 fold improvementand the binding to T2 cell revealed an excellent avidity for HLA A0201molecules (FIG. 1A).

Of the peptides derived from b2a2, all were generated from a peptidethat does not predict a good binding to HLA A0201. Three peptides, b2a2A3-A5 (Table 1) bound well to HLA A0201 molecules (FIG. 1B). These threepeptides have a tyrosine-leucine sequence substitution at position 1 and2 and also a valine substitution in position 6 or 9.

Example 10 Assessment of the Dissociation Time of b2a2 and b3a2Synthetic Peptides Analogues from HLA A020J

The immunogenicity of peptide antigens depends on a low dissociationrate of MHC/peptide complexes. The stability of complexes formed betweenHLA-A0201 and the b3a2 analogue peptides was assayed on T2 cells overtime. Overnight incubation of T2 cells with saturating amounts ofHLA-A0201 binding peptides and human β2 microglobulin resulted inincreased surface expression of HLA-A0201 molecules. After peptideremoval and addition of Brefeldin A to inhibit protein synthesis, T2cells were incubated at 37° C. and the amount of HLA-A0201 moleculesremaining at the cell surface was determined after various incubationtimes.

The stability of each peptide/HLA-A0201 complex was then normalizedrelative to that observed for the tyrosinase D peptide or HIV gagpeptide which are peptides with known high affinity and half life.HLA-A0201 complexes formed with peptides p210A and p210B were unstable,reaching background levels in less than 1 h of 5 incubation at 37° C. Incontrast, peptides p210C, p210D, p210E and p210F formed complexes thatwere relatively stable over 6-8 hours.

Example 11 Induction of CD8 Immune Response Against b2a2 and b3a2Synthetic Peptide Analogues

While affinity for MHC molecules is necessary for peptideimmunogenicity, there is also a requirement for presence of reactiveprecursor T cells with appropriate T cell receptors. Using an optimizedT cell-expansion system, with monocyte derived DC, CD 14+ cells as APC,and purified T cells, the ability of the synthetic b3a2 and b2a2analogues to stimulate peptide-specific CTLs is examined. Ten healthyHLA A0201 donors as well 5 patients with CML were studied. Five out ofthe ten individuals responded to immunization, generating T cells thatsecreted IFN gamma when challenged with different peptide-pulsed T2cells as targets. p210D and p210E produced an immune response in some ofthe donors tested although p210C and p210F generated a more consistentand higher immune-responses (FIG. 2A), Responses were observed after thesecond or third round of peptide stimulation after CD8⁺ isolation or inunpurified CD3⁺T cells.

The spot numbers were consistently higher with peptides that bound withhigher affinity to HLA 0201 molecules as determined by T2 assay. Moreimportantly, T cells generated in the presence of the new syntheticanalogues were able to recognize the native sequences. p210C and p210Fwere able to stimulate T cells to recognize their respective nativesequences (FIG. 2A). CML A2, the native sequence from p210F is a naturalweak binder and there is indirect evidence that it can be naturallyexpressed in the surface of chronic myelogenous leukemia blasts. Noimmune response could be generated against the p210A and p210B, despiteattempts using different donors. This result is consistent with theirreduced affinity for MHC.

A chronic myelogenous leukemia patient in chronic phase HLA A0201responded to p210C stimulation of T cells and demonstrated T-cellcross-reactivity with native p210cn peptide. Response was observed afterthe second round of T-cell stimulation in vitro (FIG. 2B).

The peptides derived from b2a2 also generated a significant immuneresponse as measured by gamma interferon secretion CD3+T cells. Peptidesb2a2A3, A4 and A5 generated an immune response in two healthy donors(FIG. 3A). The response against b2a2 A3 was more consistent betweendonors. T cells generated in the presence of b2a2 A3 were able toidentify the original native sequence. This is of special relevancebecause the native sequence is a weak/intermediate binder to HLA. Again,a CML patient in chronic phase HLA A0201 responded to b2a2 A3stimulation of T cells and demonstrated T-cell cross-reactivity withnative b2a2 A peptide (FIG. 3B).

Gamma interferon ELISPOT is not always associated with functionalkilling. Therefore the T cell lines obtained after several stimulationswith the analogue peptide were tested in a classic chromium-51 assayusing peptide pulsed target cell lines. T cells generated in vitro inthe presence of p210C (FIG. 4A) and b2a2 A3 (FIG. 4B) were able to killT2 cell line pulsed with specific peptides but not without peptide orwith control peptide, This experiment was also performed using HLAmatched chronic myelogenous leukemia cell lines or CML blasts expressingthe respective translocation b.3a.2 or b2a2. Significant cytotoxicitywas generated raising the possibility that the native peptides were notnaturally processed and/or sufficiently expressed in the surface of theleukemic cells.

Example 12 Binding of HLA-A0201 and -A030 I by Synthetic PeptideAnalogues Derived from the WT1 Oncoprotein

Thermostabilization assays using a TAP 1/2 negative cell line (T2) and amodified protocol using Raji A0301 cells showed that several peptidesthat were predicted to be good binders to HLA A0201 or A0301 molecules,could stabilize MHC class I A0201 or A0301 molecules (Table 2), Thesynthetic analogues WT1-A1, -B1, C1, and -D1 all predicted to bind HLAA0201 better than the respective native WT-1 peptides demonstratedsimilar or increased binding compared to WT1-A, -B, C, and D (FIG. 5A).WT1-D1 demonstrated a significantly higher level of binding to HLA-A0201over WT1-D which was similar to control. A comparison of HLA A0301binding of A3 WT1-A, -B, -C, and -D with each of their respective threeanalogues demonstrated relatively similarly binding (FIGS. 5B-5E).

Example 13 Induction of CDS or CD3 Immune Response Against SyntheticPeptide Analogues Derived from the WT1 Oncoprotein

Cells were primarily stimulated with autologous monocyte-derived,peptide-pulsed dendritic cells generated in the presence of GM-CSF,IL-4, TNF alpha, PGE2 and CD40L and re-stimulated with peptide-pulsed CD14⁺ monocytes in the presence of IL-2 and IL-7. After two to fourstimulations, the CD8⁺CTL lines were assessed by either IFN alphaELISPOT or a chromium release assay using pulsed, HLA-matched leukemiccell lines (FIGS. 6A-6B). Several analogue peptides generated greaterimmune responses, i.e., increased CD8 T cell precursor frequency, incomparison with the native peptides (FIGS. 7A-7D). CD8⁺T cellsstimulated with the new synthetic peptides cross-reacted with the nativeWT1 peptide sequence and are able to kill HLA matched chronicmyelogenous leukemia blasts (FIGS. 8A-8B)

The following references are cited herein:

-   1. Kessler et al. J Exp Med, 185(4): p. 629-40 (1997).-   2. Dyall et al, J Exp Med, 188(9): p. 1553-61 (1998).-   3. Valmori et al, J Immunol, 165(1): p. 5.3.3-8 (2000).-   4. Valmori et al 0.1 Immunol, 164(2): p. 1125-31 (2000).

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was incorporated specifically and individually byreference. One skilled in the art will readily appreciate that thepresent invention is well adapted to carry out the objects and obtainthe ends and advantages mentioned, as well as those inherent therein.The present examples along with the methods, procedures, treatments,molecules, and specific compounds described herein are presentlyrepresentative of preferred embodiments, are exemplary, and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art which areencompassed within the spirit of the invention as defined by the scopeof the claims.

1. A synthetic WT-1 peptide comprising a sequence of amino acidsselected from the group consisting of YMFPNAPYL (SEQ ID NO: 18),YLGEQQYSV (SEQ ID NO: 20), YLLPAVPSL (SEQ ID NO: 22), YLGATLKGV (SEQ IDNO: 24), YLNALLPAV (SEQ ID NO: 26), GLRRGIQDV (SEQ ID NO: 28), KLYFKLSHL(SEQ ID NO: 30), ALLLRTPYV (SEQ ID NO: 32), YMTWNQMNL (SEQ ID NO: 34),NMYQRNMTK (SEQ ID NO: 36), NMHQRVMTK (SEQ ID NO: 37), NMYQRVMTK (SEQ IDNO: 38), QMYLGATLK (SEQ ID NO: 40), QMNLGVTLK (SEQ ID NO: 41), QMYLGVTLK(SEQ ID NO: 42), FMYAYPGCNK (SEQ ID NO: 44), FMCAYPFCNK (SEQ ID NO: 45),FMYAYPFCNK (SEQ ID NO: 46), KLYHLQMHSR (SEQ ID NO: 48), KLSHLQMHSK (SEQID NO: 49), and KLYHLQMHSK (SEQ ID NO: 50), that is an analogue peptideof a native WT-1 peptide selected from the group consisting of RMFPNAPYL(SEQ ID NO: 17), SLGEQQYSV (SEQ ID NO: 19), ALLPAVPSL (SEQ ID NO: 21),NLGATLKGV (SEQ ID NO: 23), DLNALLPAV (SEQ ID NO: 25), GVFRGIQDV (SEQ IDNO: 27), KRYFKLSHL (SEQ ID NO: 29), ALLLRTPYS (SEQ ID NO: 31), CMTWNQMNL(SEQ ID NO: 33), NMHQRNMTK (SEQ ID NO: 35), QMNLGATLK (SEQ ID NO: 39),FMCAYPGCNK (SEQ ID NO: 43), and KLSHLQMHSR (SEQ ID NO: 47), wherein saidanalogue specifically binds to HLA A0201 or HLA A0301 molecules on ahuman cell.
 2. The synthetic WT-1 peptide of claim 1, wherein saidanalogue peptide is a degradation product of said synthetic WT-1peptide.
 3. The synthetic WT-1 peptide of claim 1, further comprising:an immunogenic carrier linked thereto.
 4. The synthetic WT-1 peptide ofclaim 3, wherein said immunogenic carrier is a carrier protein, acarrier peptide or an antigen-presenting cell.
 5. The synthetic WT-1peptide of claim 4, wherein said carrier protein or carrier peptide iskeyhole limpet hemocyanin, an albumin or a polyamino acid.
 6. Thesynthetic WT-1 peptide of claim 4, wherein said antigen presenting cellis a dendritic cell.
 7. The synthetic WT-1 peptide of claim 1, wherein atotal number of amino acids in said analogue peptide is about 70% toabout 130% of a total number of amino acids in said native WT-1 peptide.8. The synthetic WT-1 peptide of claim 8, wherein said analogue peptidehas about 8 to about 12 amino acids. 9.-11. (canceled)
 12. The syntheticWT-1 peptide of claim 1, wherein said human cell is characteristic of apathophysiologic state.
 13. The synthetic WT-1 peptide of claim 12,wherein said pathophysiologic state is a cancer selected from the groupconsisting of a leukemia, a breast cancer, a lymphoma, a mesothelioma, alung cancer, a testicular cancer, and an ovarian cancer.
 14. Thesynthetic WT-1 peptide of claim 13, wherein said leukemia is a chronicmyelogenous leukemia.
 15. A synthetic WT-1 peptide having an amino acidsequence selected from the group consisting of NMYQRNMTK (SEQ ID NO:36), NMHQRVMTK (SEQ ID NO: 37), NMYQRVMTK (SEQ ID NO: 38), QMYLGATLK(SEQ ID NO: 40), 25 QMNLGVTLK (SEQ ID NO: 41), QMYLGVTLK (SEQ ID NO:42), FMYAYPGCNK (SEQ ID NO: 44), FMCAYPFCNK (SEQ ID NO: 45), FMYAYPFCNK(SEQ ID NO: 46), KLYHLQMHSR (SEQ ID NO: 48), KLSHLQMHSK (SEQ ID NO: 49),or KLYHLQMHSK (SEQ ID NO: 50).
 16. A method of inducing formation andproliferation of human cytotoxic T cells that recognize a cancer cell,wherein said cancer cell presents the native WT-1 peptide of claim 1 ona major histocompatibility complex (MHC) class I molecule thereof, saidmethod comprising contacting human immune cells in vivo or ex vivo withthe synthetic WT-1 peptide of claim 1 or DNA encoding said syntheticWT-1 peptide, whereby said synthetic WT-1 peptide or DNA encoding saidsynthetic peptide induces formation and proliferation of said humancytotoxic T cells.
 17. The method of claim 16, whereby said humancytotoxic T cells are capable of mounting a heteroclitic immune responseagainst said cancer cell.
 18. The method of claim 16, wherein the stepof contacting said human immune cells are is performed in vivo in anindividual having a cancer.
 19. A method of treating a subject having acancer, wherein a malignant cell of said cancer presents the native WT-1peptide of claim 16 on a major histocompatibility complex (MHC) class Imolecule thereof, comprising a. inducing in vivo in a donor or ex vivousing immune cells from a donor formation and proliferation of humancytotoxic T cells that recognize said malignant cell by the method ofclaim 16; and b. infusing said human cytotoxic T cells into saidsubject, thereby treating a subject having a cancer. 20.-21. (canceled)22. The method of claim 16, whereby said DNA molecule is in a vector oran antigen presenting cell.
 23. The method of claim 16, wherein saidhuman immune cells are peripheral blood mononuclear cells, bone marrowcells, dendritic cells, or macrophages.
 24. The method of claim 16,wherein said cancer cell is selected from the group consisting of aleukemia cell, a breast cancer cell, a lymphoma cell, a mesotheliomacell, a lung cancer cell, a testicular cancer cell, and an ovariancancer cell. 25.-29. (canceled)
 30. A pharmaceutical composition,comprising: a therapeutically effective amount of the synthetic WT-1peptide of claim 1 or a DNA encoding said synthetic WT-1 peptide; and apharmaceutically acceptable carrier.
 31. The pharmaceutical compositionof claim 30, wherein said DNA is in a vector or an antigen presentingcell. 32.-33. (canceled)
 34. The pharmaceutical composition of claim 30,further comprising an adjuvant, a diluent or a combination thereof. 35.The pharmaceutical composition of claim 34, wherein said adjuvant is aprotein, a peptide or an antigen presenting cell linked to saidsynthetic peptide.
 36. The pharmaceutical composition of claim 35,wherein said protein or peptide is keyhole limpet hemocyanin, an albuminor a polyamino acid.
 37. The pharmaceutical composition of claim 35,wherein said antigen presenting cell is a dendritic cell. 38.-42.(canceled)
 43. The method of claim 16 wherein said synthetic WT-1peptide further comprises an adjuvant.